The lipid A moiety of lipopolysaccharide (LPS) forms the outer monolayer of the outer membrane of most Gram-negative bacteria. Escherichia coli lipid A is synthesized on the cytoplasmic surface of the inner membrane by a conserved pathway of nine constitutive enzymes, which were discovered by the PI. Following the assembly of the core oligosaccharide, the core-lipid A complex is flipped to the outer surface of the inner membrane by the ABC transporter MsbA, where the O-antigen polymer is attached. Subsequent trafficking of nascent LPS to the outer membrane involves a second ABC transporter and two outer membrane proteins, encoded by the lpt genes. Many additional covalent modifications of lipid A may occur during its transit from the outer surface of the inner membrane to the outer membrane. Lipid A modification enzymes are therefore excellent reporters for LPS trafficking within the bacterial cell envelope. However, modification systems are quite variable between different Gram-negative organisms and are often regulated by environmental factors. Although not required for growth under laboratory conditions, the lipid A modification enzymes can modulate the virulence of some pathogens. The specific aims for the coming grant period deal mainly with the constitutive enzymes. They are: 1) mechanistic and structural studies of the acyltransferases LpxA and LpxD; 2) the characterization of E. coli mutants resistant to LpxC inhibitors; 3) biochemical and structural studies of enzymes that process UDP- diacylglucosamine; 4) the characterization of novel lipid A biosynthetic enzymes in bacteria lacking LpxK, KdtA and LpxL; and 5) the development of in vitro systems for analyzing LPS core assembly and LPS trafficking in E. coli. A complete understanding of the structures and mechanisms of the lipid A biosynthetic enzymes will enable the discovery of new inhibitors with antibiotic activity against multi-drug resistant Gram-negative bacteria. Mutation or heterologous expression of the genes encoding the lipid A biosynthetic and modification enzymes will facilitate the re-engineering of lipid A structure in diverse bacteria and may enable the development of new vaccines. A complete understanding of LPS biosynthesis, modification, and trafficking will also provide fundamental insights into outer membrane biogenesis and function. PUBLIC HEALTH RELEVANCE: Half of all human bacterial pathogens are classified as Gram-negative; they appear as pale, pink rods in the light microscope because of their inability to take up a purple dye, called Gram-stain. Bacteria of this kind, which include all strains of Escherichia coli, Salmonella, Pseudomonas, and Francisella, contain an outer membrane that makes them impermeable to certain dyes and antibiotics. The outer surfaces of the outer membranes of Gram-negative bacteria contain large amounts of a unique substance known as lipopolysaccharide (LPS), which is held in place by lipid A. The enzymes that assemble the lipid A anchor of LPS are conserved and are excellent targets for designing new antibiotics with activity against Gram-negative pathogens that have become resistant to commercial antibiotics. In addition, the modification of lipid A structure in live bacteria by manipulation of the genes encoding the enzymes that assemble the lipid A domain of LPS are useful for attenuating bacterial pathogens so that they can be used as vaccines.